Naturally occurring membrane channels and pores are formed from a large family of diverse proteins, peptides and organic secondary metabolites whose vital biological functions include control of ion flow, signal transduction, molecular transport and production of cellular toxins. (Eisenberg, B. (1998) Ionic channels in biological membranes: natural nanotubes. Acc. Chem. Res. 31:117; Hill, B. (1992) Ionic Channels of Excitable Membranes 2nd edn. (Sinauer Associates, Sunderland); Gennis, R. B. (1989) Biomembranes, Molecular Structure and Function, Springer, New York). Many pore-forming peptides, such as gramicidin and alamethicin, function by creating pores within the plasma membrane of a target cell (Marsh, D. (1996) Peptide models for membrane channels. Biochem. J. 315(pt2):345; Smart, O. S.; Goodfellow, J. M.; Wallace, B. A. (1993) The pore dimensions of gramicidin A. Biophys. J. 65(6):2455; Ritov, V. B.: Tverdislova, I. L.; Avakyan, T. Yu; Menshikova, E. V.; Leikin, Yu N.; Bratkovskaya, L. B.; Shimon, R. G. (1992) Alamethicin-induced pore formation in biological membranes. Gen. Physiol. Biophys. 11(1):49). Pore-forming protein toxins, such as the Staphylococcus aureus xcex1-hemolysin and Streptococcus streptolysin-O, act similarly by boring holes into the cell membranes. (Bayley, H. (1997) Toxin structure: part of a hole? Curr. Biol. 7(12):R763; Ikigai, H.; Nakae, T. (1987) Assembly of the alpha-toxin-hexame of Staphylococcus aureus in the liposome membrane. J. Biol. Chem 262:2156; Palmer, M; Vulicevic I.; Saweljew, P.; Valeva, A.; Kehoe, M.; Bhakdi, S. (1998) Biochem. 37(8):2378. Streptolysin-O: a proposed model of allosteric interaction between a pore-forming protein and its target lipid bilayer. xcex1-Hemolysin has received intense interest as a prototype for artificial molecular gatekeepers that can be used for the design of drugs, (Bayley, H. (1997) Building doors into cells. Sci. Am. 277 (September):62); (Panchal, R. G.; Cusak, E.; Cheley, S.; Bayley, H. (1996) Turnor-protease-activated, pore-forming toxins from a combinatorial library, Nature Biotechnol 14:852) drug delivery agents (Fernandex, T.; Bayley, H. (1998) Ferrying proteins to the other side. Nature Biotechnol. 16(5):418) or highly sensitive and selective biosensors. (Braha, O.; Walker, B.; Cheley, S.; Kasianowicz, J. J.; Song, L.: Gouaux, J. E.; Bayley, H. (1997) Designed protein pores as components for biosensors. Chem. Biol. 4:497). Difficulties associated with using protein molecules in these designs include heat and mechanical instability, immunogenicity in biotherapeutics, and the like.
Lying at the center of the pore assembled from seven molecules of xcex1-hemolysin (and many other pore-forming proteins) is a nanosize channel. (Song, L; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J. E. (1996) Structure of Staphylococcal xcex1-hemolysin, a heptameric transmembrane pore. Science 274:1859). The transmembrane segment, a xcex2-barrel, of the channel ranges from 14 xc3x85 to 26 xc3x85 in diameter and 52 xc3x85 in length. The interior of the xcex2-barrel was found to be primarily hydrophilic, while the exterior has a hydrophobic belt.
Despite the existence of numerous chemical models as artificial transmembrane channels, (Alkerfeldt, K. S.; Lear, J. D.; Wasserman, Z. R.; Chung, L. A.; DeGrado, W. F. (1993) Acc. Chem. Res. 26:191; Gokel, G. W.; Murillo, O. (1996) Acc. Chem. Res. 29:425) the design and synthesis of artificial systems that can mimic the biological function of natural compounds is still a formidable task. A successful model rivaling the structural robustness and versatility as observed in the natural systems has not been seen until the present invention. Such a model requires a tube-or barrel-like structure with a nanosized, hydrophilic internal cavity and a hydrophilic internal cavity and a hydrophobic outside surface.
The self-assembly of cyclic peptides (Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.; Khazanovich, H. (1993) Self-assembling organic nanotubes based on a cyclic peptide architecture. Nature 366:324) provides an example of self-assembling nanotubes. However, Ghadiri""s nanotubes do not suit the above purpose since these tubes consist of stacked peptide rings. Due to the structure of these cyclic peptides and the way the tubes form, it is difficult to imagine how different molecular switches could be put site-specifically into these nanotubes.
Other approaches have been described (Bryson, J. W.; Betz, S. F; Lu, H. S.; Suich, D. J.; Zhou, H. X.; O""Neil, K. T.; DeGrado, W. F. (1995) Protein design: a hierarchic approach. Science 270:935) toward manipulating nanoscale structures by designing oligomeric bundles of xcex1-helices. Models for transmembrane helical oligomers may lead to simplified systems for designing pore-forming agents. (Dieckmann, G. R.; DeGrado, W. F. (1997) Modeling transmembrane helical oligomers Curr. Opin. Struct. Biol. 7(4):486). The advantage of these helix bundles is that they allow precise control over the positions to be modified, enabling site-specific engineering of the nanostructures with both natural and unnatural amino acids. However, one disadvantage of these designed helix bundles is that they may have the same instability and immunogenicity problems associated with natural peptides and proteins.
Until the present invention, a question that still remained was whether unnatural systems provide nanosize, tube-like structures. Various prior art approaches to folding structures have been taken, involving primarily the use of intramolecular hydrogen bonding, or donor-acceptor interactions. (Gellman, S. H. (1998) Foldamers: a manifesto. Acc. Chem. Res. 31:173) Examples of folded, potentially functionalizable structures include xcex2-peptides and peptoid oligomers and many others involving unnatural backbones. (Appella, D. H; Christianson, L. A.; Karle, I. L; Powell, D. R.; Gellman, S. H. (1996) Beta-Peptide foldamers: robust helix formation in a new family of beta-amino acid oligomers. J. Am. Chem. Soc. 118:13071-13072; Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.; Oberer, L.; Hommel, U.; Widmer, H. (1996) Beta-Peptides: synthesis by Arndt-Eistert homologation with concomitant peptide coupling. Structure determination by NMR and CD spectroscopy and by X-ray crystallography. Helical secondary structure of a xcex2-hexapeptide in solution and its stability towards pepsin. Helv. Chim. Acta 79:913; Armand, P.; Kirshenbaum, K.; Falicov, A.; Dunbrack, R. L. Jr.; Dill, K. A.; Zuckermann, R. N.; Cohen, F. E. (1997) Chiral N-substituted glycines can form stable helical conformations. Fold. Des. 2(6):369; and others such as Cho, C. Y.; Moran, E. J.; Cherry, S. R.; Stephans, J. C.; Fodor, S. P.; Adams, C. L.; Sundaram, A.; Jacobs, J. W.; Schultz, P. G. (1993) An unnatural biopolymer, Science 261:1303). However, few examples have shown folded structures with cavities similar to those seen in protein molecules.
One approach toward building nanotubes involved designing oligomers that undergo polar solvent-driven folding (Nelson, J. C.; Saven, J. G.; Moore, J. S.; Wolynes, P. G. (1997) Solvophobically driven folding of nonbiological oligomers. Science 277:1793). While a helical conformation with nanosized tubular cavity was proposed for the folded structure, this system was, however, unsuitable for designing pore-forming agents since the interior is quite hydrophobic.
The assembly of well-defined protein secondary structures, such as xcex1-helix, xcex2-sheet, and turns, leads to a bewildering array of tertiary structures. (Branden, C.; Tooze, J., Introduction to Protein Structure, 2nd ed.; Garland Publishing: New York, 1998). As the first step toward developing artificial oligomers and polymers that fold like biomacromelecules, there is currently an intense interest in designing unnatural building blocks that adopt well-defined secondary structures. (Gellman, S. H., Acc. Chem. Res., 1998, 31, 173; Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H., J. Am. Chem. Soc., 1999, 121, 6206. Gong, B.; Yan, Y.; Zeng, H.; Skrzypcak-Jankun, E.; Kim, Y. W.; Zhu, J.; Ickes, H., J. Am. Chem. Soc., 1999, 121, 5607. Gin, M. S.; Yokozawa, T.; Prince, R. B.; Moore, J. S., J. Am. Chem. Soc., 1999, 121, 2643. Yang, D.; Qu, J.; Li, B.; Ng, F.-F; Wang, X.-C.; Cheung, K.-K.; Wang, D.-P; Wu, Y.-D., J. Am. Chem. Soc., 1999, 121, 589. Hanessian, S.; Luo, X.; Schaum, R.; Michnick, S., J. Am. Chem. Soc., 1998, 120, 8569. Seebach, D.; Abele, S.; Stifferlen, T.; Hanggi, M.; Gruner, S.; Seiler, P., Helv. Chim. Acta, 1998, 81, 2218. Armand, P.; Kirshenbaum, K.; Goldsmith, R. A.; Farr-Jones, S.; Barron, A. E.; Truong, K. T.; Dill, D. A.; Mierke, D. F.; Cohen, F. E.; Zuckermann, R. N.; Bradley, E. K., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 4309).
This invention relates to a novel class of compositions such as oligomers and polymers that automatically fold into helices with large (10 xc3x85 to 50 xc3x85) tubular cavities. The compositions are comprised of aromatic rings linked by amide groups. The backbone of the composition is curved due to the incorporation of intramolecular hydrogen bonds that rigidify the amide linkages. The backbone is long enough to fold back on itself, leading to a left- or right-handed helical conformation.
The helical composition is further stabilized by stacking of the aromatic rings of neighboring spiral turns. Such a backbone-based helical programming leads to helical compositions whose folded conformation is resilient toward structural variation of the side groups which, in turn, determine the outside surface properties. The interior of the helical composition features the amide-O atoms, which make the tubular cavities hydrophilic. The internal diameters of the helices are adjustable by combining meta- and para-disubstituted benzene rings or by using larger aromatic rings, such as derivatives of naphthalene and anthracene.
These helices, as nanotubes, are useful as artificial pore-forming agents which are readily functionalized. Molecular gatekeepers based on these nanotubes are designed by including biochemical, chemical, and physical switches into the structures. The present invention is especially useful for design of pore-forming drugs, drug carriers, novel chiral hosts for chiral recognition and catalysis, and sensitive membrane-bond ion-channels and sensors.
The helical compositions of present invention are also useful as pore-forming drugs, membrane-bound nanopores for DNA sequencing, and new optical materials.
The present invention also relates to methods for making nanotube compositions and the nanotube compositions themselves. These nanotube compositions mimic part or all of the pore-forming function of xcex1-hemolysin and other similar proteins. Artificial pores that behave in a controlled and predictable fashion are synthesized according to the present invention. These nanotube compositions are useful as drugs, to deliver drugs or as biosensors to detect toxic chemicals.
The compositions have a nanosized barrel- or tube-like structure with a hydrophilic interior and a hydrophobic exterior. To avoid the problems associated with protein molecules, the nanotube compositions have mechanical and heat stability for designing sensors and other materials, or are biologically inert (enzyme resistant and non-immunogenic) in biotherapeutics. To control the pore""s ability to open and close, the nanotube compositions are readily modifiable to enable molecular switches to be incorporated into one or more specific sites.
The present invention also relates to new nanotube compositions based on the folding of designed oligomers. These nanotube compositions satisfy all the requirements, as defined above, for designing pore-forming agents, for the design of various desirable functional agents.
The present invention provides well-defined, folded conformations through rigidification of backbones. The conformations have (i) electrostatically negative, hydrophilic interior cavities; (ii) modifiable exterior surfaces; and (iii) adjustable cavity size.
In one aspect of the present invention, the conformations are useful for transmembrane channels and pores.
In this aspect, the following applications are particularly useful:
(i) Synthesis of Compositions: monomers with membrane-compatible side groups (selected xcex1-amino acid side chains); sequence-specific synthesis of oligomers on solid-supports.
(ii) Characterization of Compositions: x-ray crystallography; 1D and 2D NMR; IR; facilitated by sequence information from synthesis.
(iii) Membrane studies: liposome-based proton transport; single channel conductance measurements; hydrophilic (sugar or dye) molecule transport; nanotube orientation in lipid bilayer as probed by polarized attenuated total reflectance (ATR), grazing-angle reflection-absorption, and FT-IR spectroscopy.
(iv) Gated nanotubes with physical or biochemical switches.
(v) Cytotoxicity assays (with the oligomers alone or with drug transport).
Still other applications include: (i) Channel- and pore-forming reagents (toxins), (ii) ion- and small molecule-transport (drug delivery), and (iii) Gated pore- and channels, and (controlled drug delivery and cell-based sensors).
In another aspect of the present invention, the conformations are useful as nanoporous polymers. In this aspect, the following applications are particularly useful
(i) Synthesis of Compositions: monomers with hydrophobic and cross-linkable side chains; polymerization of monomers (mostly one-pot); block copolymers with polymerization of short oligomers.
(ii) Characterization of Compositions: gel permeation chromatography; light scattering; IR and UV (intramol. H-bonds; hypochromic effects of stacked aromatic ringsxe2x80x94folding of polymers).
(iii) Formation of polymer films: by simple casting, and by solid- and liquid-phase, lateral cross-linking of polymer chains (oxidation, metathesis, and lateral polymerization).
(iv) Permeate flux and solute selectivity of the porous materials.
(v) Confinement and parallel alignment of close-packed guest molecules in bulk polymeric materials. Binding of guest molecules into the tubular cavities (probed by affinity columns and/or by fluorescence quenching); nanowires from reduction of metal ions absorbed into the tubular cavities.
Still other applications include: (i) polymer films with a single pore size: highly efficient materials for separation and purification; (ii) tubular cavities for aligning interesting chromophores: electro-optic materials with the robustness of polymers and the flexibility of host-guest interactions; and, (iii) templates for the fabrication of conducting nanowires.